2.2. Body composition using MRI (magnetic resonance imaging), food intake, indirect calorimetry and ex vivo respirometry

Body composition was measured after 32 weeks on RCD or HFD using an Echo MRI nuclear magnetic resonance system (Echo Medical Systems, Houston, TX). Food intake was quantified (over 72 hrs) at 8–10 and 30–32 weeks of age in mice housed individually in cages containing pre-weighed amounts of food. Food intake data in this paper are shown from mice maintained on diet for 30–32 weeks. After 16–18 weeks on RCD or HFD, mice were subjected to indirect calorimetry experiments to measure oxygen consumption, carbon dioxide production, respiratory exchange ratio, activity, and energy expenditure (kcal/h) using a Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus OH). For measuring GCG-induced oxygen consumption measurements, mice were fasted for ∼4 h, anesthetized with sodium pentobarbital (60 mg/kg), and placed on a heating pad for 10 mins prior to transferring them into a CLAMS chamber where they were acclimatized for ∼ 8 mins and subsequently given ip injections of vehicle control (acidified PBS) followed by GCG (0.01, 0.1, or 0.5 mg/kg, Sigma, Oakville, ON) or vehicle control followed by GCG (0.5 mg/kg, Sigma), and CL316,243 (1.0 mg kg, Sigma), a selective β-3-adrenergic receptor agonist. For basal ex vivo oxygen consumption measurements, fresh tissues were extracted from mice after CO₂ asphyxiation and immediately placed into warm (37 °C) DMEM (4.5 g/l glucose, L-glutamine, Wisent, St. Bruno, QC) buffer. Tissues were weighed (∼10–20 mg), washed in filtered respiration buffer (PBS, 0.02% fatty acid free BSA, 25 μM glucose, 0.01% (vol/vol) of 100 mM Na pyruvate (Sigma)), minced with scissors, re-suspended in respiration buffer and placed into a Mitocell chamber (MT200A, Strathkelvin Instruments, North Lanarkshire, Scotland) with a Clark electrode (Strathkelvin). For each tissue, measurements were obtained from three separate depots of equivalent size. Recordings were normalized to tissue weight. For stimulated ex vivo oxygen consumption measurements, tissues were extracted from littermate mice ∼20–30 minutes after an acute injection of either PBS or GCG (0.5 mg/kg, Sigma).